Non-covalent inhibitors of thioredoxin glutathione reductase with schistosomicidal activity in vivo

Only praziquantel is available for treating schistosomiasis, a disease affecting more than 200 million people. Praziquantel-resistant worms have been selected for in the lab and low cure rates from mass drug administration programs suggest that resistance is evolving in the field. Thioredoxin glutathione reductase (TGR) is essential for schistosome survival and a validated drug target. TGR inhibitors identified to date are irreversible and/or covalent inhibitors with unacceptable off-target effects. In this work, we identify noncovalent TGR inhibitors with efficacy against schistosome infections in mice, meeting the criteria for lead progression indicated by WHO. Comparisons with previous in vivo studies with praziquantel suggests that these inhibitors outperform the drug of choice for schistosomiasis against juvenile worms.

All reactions were carried out under inert atmosphere of nitrogen and monitored by thin-layer chromatography with silica gel 60 F254 precoated glass plates. Visualization of TLC plates was performed by UV light irradiation (254 nm) or staining with phosphomolybdic acid. All reagents were purchased from commercial suppliers and were used without further purification.
Chromatographic purifications were performed using an HPFC Biotage Isolera TM Four 3.0 system using prepacked flash chromatography cartridges in normal phase (irregular silica, 40-60 µm; hexanes/ethyl acetate gradient) or reverse phase (Biotage KP-C18-HS, water/methanol gradient) modes with UV detection at 254 and 280 nm. 1 H NMR spectra were recorded on Bruker spectrometer at 400 MHz. 13 C NMR spectra were recorded on Bruker spectrometer at 100 MHz. Chemical shifts were reported in parts per million (ppm) and calibrated with CDCl3 residual peak. Coupling constants were reported in Hz and the standard abbreviations indicating multiplicity were used as follows: s = singlet, bs = broad singlet, d = doublet, t = triplet, q = quartet, and m = multiplet. Chromatographic purities of the final compounds were determined using a Shimadzu HPLC system equipped with CN-propyl column (NUCLEODUR 100-3 CN-RP, 2´50 mm, 3 µm) utilizing water/acetonitrile=90/10+0.1% formic acid eluent system for phase A and methanol+0.1% formic acid for phase B. Purities of all final products were found to be superior to 95% and determined by integration of the chromatogram after subtraction of the background using Labsolutions LCMS software at wavelengths giving the maximum absorbance. High-resolution mass-spectra (HRMS) were performed on Waters Synapt G2-Si 1M HCl in acetic acid (2 eq) was added to the mixture of aryl aldehyde (1 eq) and 2,3butanedione monoxime (1.1 eq) under cooling with ice-water. The resultant mixture was stirred for 12 hrs at ambient temperature. Diethyl ether was then added to the reaction to precipitate the product and the resultant slurry was stirred for 30 min. The precipitate was filtered and washed with ether three times. The cake was suspended in methylene chloride/water and conc. NH4OH was added to adjust pH of aqueous layer to 8. The resulting mixture was stirred for 20 min and aqueous layer was removed. Organic phase was washed with brine, dried over Na2SO4 and the solvent was removed in vacuo to provide target oxazole N-oxides with sufficient purity. If necessary, product can be purified on silica gel using CH2Cl2/MeOH=9/1 eluent mixture.

2-(Benzo
The resulting mixture was stirred at ambient temperature for 12 hrs, then washed with water, brine and dried over Na2SO4. Solvent was evaporated and the residue was purified on reverse phase Biotage KP-C18 cartridge (water/methanol eluent) to afford the final compound.   mL acetonitrile was added K2CO3 (104 mg; 3 eq) followed by the dropwise addition of propargyl bromide (90 mg; 3 eq) and the resulting mixture was stirred at ambient temperature for 12 hrs.

HPLC data for 1-12 and PRP
Supplementary Figure 8. HPLC chromatogram for 8VP70 (10VP28).       The side-chain residues contributing to the doorstop pocket in SmTGR as found by the Cast-p calculation are highlighted in yellow. 18,19 When a residue is not conserved with respect to SmTGR, the green color is used. Side chain residues within 5 Å which are involved in the recognition of compound 9 are indicated by a "$" symbol. Overall percentages of identity with respect to SmTGR: SjTGR (91%); hTrxR1 (61%), BmTrxR-d (51%), PfTrxR (47%), hGR (37%).

Supplementary
Percentages of identity with respect to the residues forming the doorstop pocket in TGR: SjTGR